电针抗脊髓损伤的实验研究

背景:现有的研究结果证实神经生长因子在脊髓损伤后神经再生修复过程中发挥着重要作用,神经再生能力低下与损伤局部缺少支持神经元生长的微环境有关。针刺对脊髓损伤后的神经保护是否通过促进损伤脊髓组织中神经生长因子水平的提高而起作用,目前尚缺少大量分子水平的实验证实数据。目的:采用形态学、原位杂交等技术观察电针对实验性脊髓损伤大鼠运动功能、脊髓组织形态及其神经生长因子mRNA表达的影响。设计:随机对照动物实验。单位:同济大学附属第十人民医院针灸科,上海市针灸经络研究所。材料:实验于1999-01/2002-02在上海市针灸经络研究所完成。选取雄性SD大鼠15只,体质量(260±9.52)g,随机分成假手术组、损伤对照组和电针组,各5只。方法:损伤对照组和电针治疗组制备脊髓损伤模型,假手术组仅作椎板切除。术后2h,对电针治疗组给予电针治疗,取大椎、命门和L2夹脊、环跳,两组(位交替使用。针刺后接G6805型电针仪,疏波,0.8Hz,0.3～0.6mA,以肌肉有轻微的抖动为宜。1次/d,每次30min,连续治疗30d。损伤对照组和假手术组不作任何处理。采用自行改良的Tarlov评分标准和Rivlin方法分别于术后2h,30d进行运动功能测定。①改良的Tarlov评分标准:以完全瘫痪,针刺时下肢无反应到正常行走记作0～6分。②改良的Rivlin方法:斜板由两块矩形的合金板通过铰链于一端相连而成,板从水平位置(0°)起绕轴旋转,斜面角度可以测出。将大鼠头朝前,身体纵轴与斜板纵轴垂直放置,逐渐增大板与水平间的角度,直至大鼠不能保持原有位置5s时,记录这一临界角。原位杂交检测,光镜下明视野记录神经生长因子阳性细胞数并摄片。主要观察指标:脊髓损伤大鼠运动功能评分(改良的Tarlov评分)、脊髓损伤大鼠斜面测试临界角度(改良的Rivlin方法)、各组脊髓组织神经生长因子mRNA阳性细胞数的变化。结果:实验大鼠15只,全部进入结果分析。①大鼠脊髓损伤2h后,损伤对照组和电针治疗组运动功能评分和改良的Rivlin方法斜面临界角均明显下降;30d后,两组的改良的Tarlov评分运动功能评分和斜面临界角均有不同程度的恢复,但以电针组的恢复较明显,与损伤对照组比较,差异有显著性意义35.40±6.62,27.80±1.10;(35.40±6.62)°,(27.80±1.10)°,P<0.05]。②伤后30d,电针治疗组大鼠病理损害程度较损伤对照组轻,脊髓组织形态的损伤,神经生长因子mRNA阳性细胞数的增加较损伤对照组明显(28.13±1.64,12.63±1.41,P<0.01)。结论:电针可促进脊髓组织中神经生长因子mRNA的表达,可能减轻脊髓组织形态的损伤,促进脊髓损伤大鼠运动功能的恢复,因其神经生长因子mRNA的表达而起到神经保护作用。

Effect of electro-acupuncture on treatment of spinal cord injuries in rats

BACKGROUND: At present, researches have been proved that nerve growth factor (NGF) plays an important role in repairing neural regeneration after spinal cord injuries. Poor ability of neural regeneration is related to lack of microenvironment for neuronal growth at local injured site. Whether electro-acupuncture can protect nerve after spinal cord injuries through increasing NGF level in injured spinal cord or not is lack of experimental data on the view of molecule.OBJECTIVE: To observe the effect of electro-acupuncture on motor function, morphology of spinal cord and mRNA expression of NGF in rats with experimental spinal cord injuries through morphology and hybridization in situ.DESIGN: Randomized controlled animal study.SETTING: Department of Acupuncture and Moxibustion, the Tenth People's Hospital Affiliated to Tongji University; Shanghai Institute of Acupuncture and Meridian.MATERIALS: The experiment was completed in the Shanghai Institute of Acupuncture and Meridian from January 1999 to February 2002. A total of 15 male SD rats weighing (260±9.52) g were randomly divided into sham operation group, injured control group and acupuncture group with 5 in each group.METHODS: Models of spinal cord injuries were established in injured control group and electro-acupuncture group, and rats in sham operation group were just treated with resection of lamina of vertebra. Two hours after operation, rats in electro-acupuncture group were treated with electroacupuncture at Daczhui (GV14) and Mingmen (GV4), L2 Jiaji (Ex-B2-L2)and Huantiao (GB30), alternatively. Then, G6805 electro-acupuncture apparatus was used with distant wave, 0.8 Hz, 0.3-0.6 mA, and slight shake on muscle was regarded as proper response. The electro-acupuncture was performed every 30 minutes a day for 30 days. Rats in injured control group and sham operation group were not treated with any way, and motor functions 2 hours and 30 days after operation were observed with modified Tarlov scale and Rivlin method. ① Modified Tarlov scale: Pamplegia and irresponsivity of lower limbs during electro-acupuncture were scored from 0 to 6 points. ② Modified Rivlin method: Tiltboard was consisted of two rectangular alloy boards which was connected to one end through hinge.Board turned around axis at the beginning of horizontal position (0°) and the angle of ram could be measured. Rats faced towards front were put vertically as longitudinal axis of body to longitudinal axis of tiltboard.Angle was increased gradually when rats were not able to keep the primary position for 5 seconds, and the critical angle was recorded. Hybridization in situ assay: Amount of positive NGF cells was recorded under light microscope and photographs were completed at the same time.MAIN OUTCOME MEASURES: Scale of motor function (modified Tarlov score), critical angle of ram test (modified Rivlin method) and changes of amount of positive NGF mRNA cells in rats with spinal cord injuries.RESULTS: A total of 15 experimental rats were involved in the final analysis. ① Two hours after spinal cord injuries of rats in injured control group and electro-acupuncture group, score of motor function and critical angle of ram which was measured with modified Rivlin method were decreased remarkably; 30 days later, score of motor function measured with modified Tarlov scale and critical angle of ram were increased at various degrees, especially those in electro-acupuncture group were increased remarkably, and there was significant difference from those in injured control group 35.40±6.62, 27.80±1.10; (35.40±6.62)°, (27.80±1.10)°, P ＜ 0.05]. ② Thirty days after injury, pathological lesion of rats in electroacupuncture group was milder than that in injured control group, and morphological injury of spinal cord and amount of positive NGF mRNA cells were obvious as compared with those in injured control group (28.13±1.64,12.63±1.41, P ＜ 0.01).CONCLUSION: Electro-acupuncture can improve mRNA expression of NGF in spinal cord, relieve morphological injury of spinal cord, accelerate recovery of motor function of rats with spinal cord injuries, and protect nerve through mRNA expression of NGF.